1.Molecular methods for authentication of Chinese medicinal materials.
Chuanyi WANG ; Baolin GUO ; Peigen XIAO
China Journal of Chinese Materia Medica 2011;36(3):237-242
The resource authentication is required for quality assurance and control of Chinese medicine. This review provides an informative introduction to molecular methods used for authentication of Chinese medicinal materials. The technical features of the methods based on sequencing, polymerase chain reaction (PCR) and hybridization are described, merits and demerits and development of the molecular methods in identification of Chinese medicinal materials are discussed.
Biometric Identification
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methods
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Medicine, Chinese Traditional
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standards
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Nucleic Acid Hybridization
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Polymerase Chain Reaction
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Sequence Analysis, DNA
2.Selection of reference genes of Siraitia grosvenorii by real-time PCR.
Dong-ping TU ; Chang-ming MO ; Xiao-jun MA ; Huan ZHAO ; Qi TANG ; Jie HUANG ; Li-mei PAN ; Rong-chang WEI
China Journal of Chinese Materia Medica 2015;40(2):204-209
Siraitia grosvenorii is a traditional Chinese medicine also as edible food. This study selected six candidate reference genes by real-time quantitative PCR, the expression stability of the candidate reference genes in the different samples was analyzed by using the software and methods of geNorm, NormFinder, BestKeeper, Delta CT method and RefFinder, reference genes for S. grosvenorii were selected for the first time. The results showed that 18SrRNA expressed most stable in all samples, was the best reference gene in the genetic analysis. The study has a guiding role for the analysis of gene expression using qRT-PCR methods, providing a suitable reference genes to ensure the results in the study on differential expressed gene in synthesis and biological pathways, also other genes of S. grosvenorii.
Cucurbitaceae
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genetics
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RNA, Ribosomal, 18S
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genetics
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Real-Time Polymerase Chain Reaction
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methods
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Reference Standards
3.Establishment and evaluation of multiplex PCR for detection of main pathogenic bacteria of endometritis in Tibetan sheep.
Jinhui HAN ; Meng WANG ; Yangyang PAN ; Xuequan HU ; Xingyun ZHANG ; Yan CUI ; Gengquan XU ; Libin WANG ; Sijiu YU
Chinese Journal of Biotechnology 2020;36(5):908-919
A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.
Animals
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Bacteria
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genetics
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isolation & purification
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Bacteriological Techniques
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methods
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Endometritis
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microbiology
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veterinary
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Female
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Multiplex Polymerase Chain Reaction
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standards
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Polymerase Chain Reaction
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veterinary
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Sensitivity and Specificity
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Sheep
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Sheep Diseases
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microbiology
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Tibet
4.Development of A synthetic positive control for the nucleic acid detection of mumps virus.
Ai-li CUI ; Li JIN ; Wen-bo XU
Chinese Journal of Virology 2013;29(5):544-547
To rapidly identify the cross-contamination problems caused by the positive control in the process of mumps virus nucleic acid detection, a new mumps virus RNA positive control was developed in this study. Using the same primers and reaction conditions, the cross-contamination problems caused by the positive control could be readily identified by comparing the fragments lengths of the PCR products between the positive control and the samples. This new RNA positive control of mumps virus can be widely used in the diagnosis and genotyping of mumps virus as a better laboratory quality control.
Cell Line
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DNA Primers
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genetics
;
standards
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Genotype
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Humans
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Mumps
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diagnosis
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virology
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Mumps virus
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genetics
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isolation & purification
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RNA, Viral
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genetics
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Reference Standards
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Reverse Transcriptase Polymerase Chain Reaction
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methods
;
standards
5.Assessment of comparative genomic hybridization experiment by an in situ synthesized CombiMatrix microarray with Yersinia pestis vaccine strain EV76 DNA.
Yuan-Hai YOU ; Peng WANG ; Yan-Hua WANG ; Hai-Bin WANG ; Dong-Zheng YU ; Rong HAI ; Jian-Zhong ZHANG
Biomedical and Environmental Sciences 2010;23(5):384-390
OBJECTIVEThe quality of microarray data influences the accuracy of comparative genomic analyses to a large extent. To ensure that the results obtained by using an in situ synthesized microarray are accurate, data quality is to be assessed by evaluating the melting temperature (Tm) of probes, probability of false synthesis rates, and fragmentation of labeled targets.
METHODSDNA from the Yersinia pestis vaccine strain EV76 was used for microarray analyses. Microarray results were confirmed by PCR. Statistical and bioinformatics methods were employed to perform microarray data analyses and evaluation.
RESULTSCorrelation coefficients of the three datasets were above 0.95 after two-time stripping and hybridization with a labeled DNA with the size of fragmentation being 200 bp - 2 kb, which showed that the hybridization results were highly reproducible. Correlation coefficients were lower with the values ranging from 0.87 to 0.92 between the datasets generated from hybridization with different sizes of the labeled DNA fragment. For the relationship between Tm and signal intensity, there was a different distribution of Tm in the lowest 300 or 3,000 probes with a range of 70 °C-72 °C and the highest 300 or 3,000 probes with a range of 72 °C-74 °C.
CONCLUSIONThe results of this study suggest that the initial microarray design may affect the accuracy of final analyses and that the probe Tm and the size of the labeled fragment may be the two factors of the greatest importance.
Cluster Analysis ; Comparative Genomic Hybridization ; methods ; standards ; DNA Fragmentation ; DNA, Bacterial ; genetics ; Genome, Bacterial ; Oligonucleotide Array Sequence Analysis ; methods ; standards ; Polymerase Chain Reaction ; Reproducibility of Results ; Yersinia pestis ; genetics
6.Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation.
Yousry HAWASH ; M M GHONAIM ; Ayman S AL-HAZMI
The Korean Journal of Parasitology 2015;53(2):147-154
Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (approximately375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects approximately550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, approximately2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.
Cryptosporidiosis/*diagnosis/*parasitology
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Cryptosporidium/genetics/*isolation & purification
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DNA Primers/genetics
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DNA, Protozoan/genetics
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Feces/parasitology
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Humans
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Polymerase Chain Reaction/methods/*standards
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Reference Standards
7.Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.
Dan CHEN ; Shiyang PAN ; Erfu XIE ; Li GAO ; Huaguo XU ; Wenying XIA ; Ting XU ; Peijun HUANG
Annals of Laboratory Medicine 2017;37(1):18-27
BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.
Adult
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DNA/*blood/standards
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Female
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Healthy Volunteers
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Humans
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Male
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Middle Aged
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Real-Time Polymerase Chain Reaction/*methods/standards
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Reference Values
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Wounds and Injuries/blood
8.Stability analysis of reference gene based on real-time PCR in Artemisia annua under cadmium treatment.
Liang-Yun ZHOU ; Ge MO ; Sheng WANG ; Jin-Fu TANG ; Hong YUE ; Lu-Qi HUANG ; Ai-Juan SHAO ; Lan-Ping GUO
China Journal of Chinese Materia Medica 2014;39(5):777-784
In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.
Artemisia annua
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drug effects
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genetics
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metabolism
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Cadmium
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pharmacology
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Plant Leaves
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drug effects
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genetics
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metabolism
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Plant Proteins
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genetics
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metabolism
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Real-Time Polymerase Chain Reaction
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methods
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standards
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Reference Standards
9.Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis.
Ankush RAJ ; Netrapal SINGH ; Krishna B GUPTA ; Dhruva CHAUDHARY ; Aparna YADAV ; Anil CHAUDHARY ; Kshitij AGARWAL ; Mandira VARMA-BASIL ; Rajendra PRASAD ; Gopal K KHULLER ; Promod K MEHTA
Yonsei Medical Journal 2016;57(1):88-96
PURPOSE: Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. MATERIALS AND METHODS: We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. RESULTS: Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. CONCLUSION: These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.
Bacteriological Techniques/methods
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DNA Transposable Elements/genetics
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DNA, Bacterial/analysis/genetics
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Early Diagnosis
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Female
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Gene Amplification
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Humans
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Male
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Multiplex Polymerase Chain Reaction/*methods
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Mycobacterium tuberculosis/genetics/*isolation & purification
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Polymerase Chain Reaction/*methods/standards
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Sensitivity and Specificity
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Tuberculosis/*diagnosis
10.Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii.
Meng-Ting LUO ; Jun-Zhang QUBIE ; Ming-Kang FENG ; A-Xiang QUBIE ; Bin HE ; Yue-Bu HAILAI ; Wen-Bing LI ; Zheng-Ming YANG ; Ying LI ; Xin-Jia YAN ; Yuan LIU ; Shao-Shan ZHANG
China Journal of Chinese Materia Medica 2023;48(21):5759-5766
Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.
Real-Time Polymerase Chain Reaction/methods*
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Paeonia/genetics*
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Actins/genetics*
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Reproducibility of Results
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Transcriptome
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Glyceraldehyde-3-Phosphate Dehydrogenases/genetics*
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Reference Standards
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Gene Expression Profiling/methods*